PROJECT SUMMARY Streptococcus pneumoniae (SPN) is the primary causative agent for community-acquired pneumonia in the United States and Europe and remains the leading cause of bacterial pneumonia and meningitis in children worldwide, making it a ?major global public health problem? according to the World Health Organization. While current vaccines target surface polysaccharides that comprise only a subset of the known serotypes, strategies aimed against a more widely expressed protein virulence factor have been advocated, such as the SPN IgA1 Protease (IgA1P) under investigation here. However, the relatively large size of SPN IgA1P and its IgA1 substrate that it cleaves to thwart the initial host immune response has previously precluded studies aimed at elucidating its molecular structure and interactions. By developing novel strategies through an integrative approach that combines multiple biophysical/biochemical methods including cryo-EM, we have begun elucidating the structure of SPN IgA1P. Remarkably, despite the presence of novel structural folds, the SPN IgA1P active site that is formed between domains is identical to other Zn-metalloproteases and confirms our previously published catalytic mechanism. Considering recent success in utilizing structure-based strategies for the development of broad-based vaccines, our goals here are to provide the first high-resolution structures of IgA1P alone, together with its IgA1 substrate, and with a first-generation monoclonal antibody (mAb) that blocks IgA1P activity. These studies will have a major impact on how we will generate vaccines and potentially therapeutics to block SPN infection through targeting IgA1P. Hypothesis: The large SPN IgA1P comprises multiple independently folded subunits that forms a unique 3- dimensional structure, which includes subunits that join to form the metalloprotease active site to properly orient the IgA1 for hinge cleavage. Furthermore, this active site is occluded by a neutralizing mAb. We will address this hypothesis through the following specific aims: Aim 1) Complete the cryo-EM structure of the novel IgA1P catalytic region to determine how its subunits interact to form the active metalloprotease. Aim 2) Determine how IgA1P interacts with its IgA1 substrate and how this interaction is blocked by a monoclonal antibody.